Báo cáo khoa học: Methylene analogues of adenosine 5¢-tetraphosphate Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases pot

Methylene analogues of adenosine 5¢-tetraphosphate Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases Andrzej Guranowski 1 ,El _ zbieta Starzyn ´ ska 1 , Małgorzata Pietrowska-Borek 1 , Jacek Jemielity 2 , Joanna Kowalska 2 , Edward Darzynkiewicz 2 , Mark J. Thompson 3 and G. Michael Blackburn 3 1 Department of Biochemistry and Biotechnology, Agricultural University, Poznan ´ , Poland 2 Department of Biophysics, Institute of Experimental Physics, Warsaw University, Poland 3 Department of Chemistry, Krebs Institute, University of Sheffield, UK Keywords adenosine 5¢-tetraphosphate; p 4 A; methylene analogues of p 4 A; nucleoside tetraphosphatase; dinucleoside tetraphosphatase Correspondence A. Guranowski, Katedra Biochemii i Biotechnologii, Akademia Rolnicza ul. Wołyn ´ ska 35, 60–637 Poznan ´ , Poland Fax: +48 61 8487146 Tel: +48 61 8487201 E-mail: guranow@au.poznan.pl Website: http://www.au.poznan.pl Note This study is dedicated to Professor Wojciech J. Stec on the occasion of his 65th birthday. (Received 9 November 2005, revised 15 December 2005, accepted 21 December 2005) doi:10.1111/j.1742-4658.2006.05115.x Adenosine 5¢-polyphosphates have been identified in vitro, as products of certain enzymatic reactions, and in vivo. Although the biological role of these compounds is not known, there exist highly specific hydrolases that degrade nucleoside 5¢-polyphosphates into the corresponding nucleoside 5¢-triphos- phates. One approach to understanding the mechanism and function of these enzymes is through the use of specifically designed phosphonate analogues. We synthesized novel nucleotides: a,b-methylene-adenosine 5¢-tetraphosphate (pppCH 2 pA), b,c-methylene-adenosine 5¢-tetraphosphate (ppCH 2 ppA), c,d-methylene-adenosine 5¢-tetraphosphate (pCH 2 pppA), ab,cd-bismethylene-adenosine 5¢-tetraphosphate (pCH 2 ppCH 2 pA), ab, bc-bismethylene-adenosine 5¢-tetraphosphate (ppCH 2 pCH 2 pA) and bc, cd-bis(dichloro)methylene-adenosine 5¢-tetraphosphate (pCCl 2 pCCl 2 ppA), and tested them as potential substrates and ⁄ or inhibitors of three specific nu- cleoside tetraphosphatases. In addition, we employed these p 4 A analogues with two asymmetrically and one symmetrically acting dinucleoside tetra- phosphatases. Of the six analogues, only pppCH 2 pA is a substrate of the two nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and human placenta, and also of the yeast exopolyphosphatase (EC 3.6.1.11). Surprisingly, none of the six analogues inhibited these p 4 A-hydrolysing enzymes. By contrast, the analogues strongly inhibit the (asymmetrical) dinu- cleoside tetraphosphatases (EC 3.6.1.17) from human and the narrow-leafed lupin. ppCH 2 ppA and pCH 2 pppA, inhibited the human enzyme with K i val- ues of 1.6 and 2.3 nm, respectively, and the lupin enzyme with K i values of 30 and 34 nm, respectively. They are thereby identified as being the strongest inhibitors ever reported for the (asymmetrical ) dinucleoside tetraphospha- tases. The three analogues having two halo ⁄ methylene bridges are much less potent inhibitors for these enzymes. These novel nucleotides should prove valuable tools for further studies on the cellular functions of mono- and di- nucleoside polyphosphates and on the enzymes involved in their metabolism. Abbreviations Ap 3 A, diadenosine 5¢,5¢¢¢-P 1 ,P 3 -triphosphate; Ap 4 A, diadenosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate; Np n N¢, dinucleoside 5¢,5¢¢¢-P 1 ,P n -polyphosphate; p 4 A, adenosine 5¢-tetraphosphate; p 5 A, adenosine 5¢-pentaphosphate; pCCl 2 pCCl 2 ppA, bc,cd-bis(dichloro)methylene-adenosine 5¢- tetraphosphate; pCH 2 ppCH 2 pA, ab,cd-bismethylene-adenosine 5¢-tetraphosphate; pCH 2 pppA, c,d-methylene-adenosine 5¢-tetraphosphate; p n N, nucleoside 5¢-polyphosphate; ppCH 2 pCH 2 pA, ab,bc-bismethylene-adenosine 5¢-tetraphosphate; pppCH 2 pA, a,b-methylene-adenosine 5¢-tetraphosphate; pppCH 2 ppA, b,c-methylene-adenosine 5¢-tetraphosphate. FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS 829 In addition to the canonical nucleoside mono-, di-, and triphosphates, cells contain various minor nucleo- tides. Among these are the nucleoside 5¢-polyphos- phates (p n Ns, where n ¼ 4), such as adenosine 5¢-tetraphosphate (p 4 A or ppppA) [1–5] and adenosine 5¢-pentaphosphate (p 5 A or pppppA) [2], and the dinu- cleoside 5¢,5¢¢¢-P 1 ,P n -polyphosphates (Np n N¢s, where N and N¢ are 5¢-O-nucleosides and n represents the num- ber of phosphate residues in the polyphosphate chain that links N and N¢ through their 5¢-positions). Typical examples are diadenosine 5¢,5¢¢¢-P 1 ,P 3 -triphosphate (Ap 3 A) and diadenosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate (Ap 4 A) [6–12]. The biological roles of these Np n N¢s are partially understood. In particular, Ap n A has been implicated in various intracellular processes [13,14] and also in extracellular signalling [15,16]. By contrast, the role of p n Ns is inadequately recognized. Almost 20 years ago, the accumulation of p 4 A and p 5 A in yeast was correlated with sporulation [2] and only recently, p 4 A was identified in human myocardial tissue and shown to modulate coronary vascular tone [4]. This compound has also been found as a constituent of the nucleotide pool present in the aqueous humour of New Zealand rabbits where it is proposed to act as a physiological regulator of intraocular pressure in the normotensive rabbit eye [5]. Enzymatic reactions that can lead to the accumula- tion of p 4 A and other p 4 Ns in cells fall into three cat- egories. The first comprises enzymes that catalyse transfer of a phosphate residue from a phosphate donor to ATP (e.g. the muscle adenylate kinase) [17]. The second category of enzymes includes those able to transfer adenylate or nucleotide residue onto tri- polyphosphates. The pA residue comes either from a mixed acyl–pA anhydride, as in the case of some li- gases and firefly luciferase [18–22], or from an enzyme–pA complex, as in the case of the DNA- and RNA-ligases [23,24]. Recently, the yeast UTP ⁄ glucose- 1-phosphate uridylyltransferase (EC 2.7.7.9) was shown to function according to the same pattern and to synthesize p 4 U by transferring the uridylyl moiety from UDP-glucose onto tripolyphosphate [25]. The third category includes several enzymes that degrade Ap 5 AorAp 6 A yielding p 4 A as one of the reaction products [26]. Degradation of p 4 A can be controlled by various nonspecific and specific p n N-degrading enzymes [26,27]. Among studies that shed light on the mechanism of the action of these phosphohydrolases are investiga- tions of the interaction of a given enzyme with its sub- strate analogues. Whereas many analogues of Ap 3 A and Ap 4 A, modified in the polyphosphate chain, in adenine(s) or in the ribose moiety(-ies), have been produced already and tested with numerous enzymes [28,29], p 4 A analogues have been synthesized only recently. ab,bc-bismethylene-p 4 A and bc,cd-bis(dichlo- ro)methylene-p 4 A were tested as agonists or antago- nists of the P2X 2 ⁄ 3 receptor [30] and a short report has appeared on the synthesis of pCH 2 pppA, pCH 2 pppG and pCH 2 pppm 7 G [31]. Here, we describe details on the synthesis of and the results of enzymatic studies on a series of novel p 4 A analogues that have a single methylene bridge substitu- ting one of the three bridging oxygens in the tetraphos- phate chain, or have two methylene bridges, or contain two dichloromethylene groups. The structures of these compounds are shown in Fig. 1. We prepared these nucleotides for evaluation first, as potential substrates and ⁄ or inhibitors of three enzymes that hydrolyse the pyrophosphate bond between the c- and d-phosphates of p 4 A and second, as inhibitors of two types of Ap 4 A hydrolase, for which p 4 A itself acts as a strong inhibitor. The p 4 A-hydrolysing enzymes are the two highly specific mononucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin (Lupinus luteus) seeds [32] and from human placenta [33], and the yeast (Saccharomyces cerevisiae) exopolyphosphatase (EC 3.6.1.11) that can hydrolyse p 4 A to ATP and phosphate [34]. The Ap 4 A hydrolases investigated are the two asymmetrically acting ones (EC 3.6.1.17), from human [35] and from narrow-leaved lupin (Lupinus angustifolius) [36] that split Ap 4 A into ATP and AMP, and the Co 2+ -dependent symmetrically acting Fig. 1. Structures of p 4 A analogues. Methylene analogues of adenosine 5¢-tetraphosphate A. Guranowski et al. 830 FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS dinucleoside tetraphosphatase (EC 3.6.1.41) that con- verts Ap 4 A into two ADPs [37]. Results and Discussion Comments on the synthesis of p 4 A analogues The preparation of intermediate ADP and ATP ana- logues followed standard methods. Their conversion into p 4 A analogues called for condensation with phos- phate (for ATP analogues), or with pyrophosphate or a methylenebisphosphonate (for ADP and ADP ana- logues). Although a variety of options were explored initially, the use of phosphoroimidazolates [31] proved to be the most reliable method and gave satisfactory yields without detailed optimization (Fig. 2). The prod- ucts were first, purified by ion-exchange chromato- graphy on DEAE-Sephadex 25A, which separates nucleotides according to net charge at pH 7.9, and readily resolved the desired products as tetra-to-penta anions from the corresponding reactants (di-to-tetra anions). Additional reverse-phase chromatography provided the product p 4 A analogues in high purity. The MS and 1 H NMR spectra of these nucleotides are unexceptional. The 31 P NMR spectra, however, pro- vide examples of ABCD spectra, whose chemical shift characteristics readily identify the nature and location of the oxygen and methylene groups bridging the four phosphorus atoms (see Supplementary material). Recognition of p 4 A analogues as substrates by the p 4 A hydrolysing enzymes Each compound was checked as a potential substrate for two highly specific nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and from human, and for the soluble exopolyphosphatase (EC 3.6.1.11) that has an inherent capacity to hydrolyse the distal pyrophosphate bond in p 4 Ns thus acting as a nucleoside tetraphosphatase. A typical reaction mixture (see Experimental procedures) contained 1 mm analogue and excess of enzyme, i.e. an amount that, under the same conditions, completely hydrolysed 1 mm p 4 Ato ATP and P i in < 15 min. Incubation was for up to 16 h and the progress of potential hydrolysis was analysed by TLC System A. Of six p 4 A analogues, only pppCH 2 pA was susceptible to hydrolysis and the relative velocities of the reactions were estimated only for the pair p 4 A ⁄ pppCH 2 pA. Figure 3 shows typical elution pat- terns of the substrate ⁄ product pairs on the reverse-phase HPLC column. Satisfactory separation of p 4 A from ATP was obtained by isocratic elution with potassium Fig. 2. Chemical synthesis of pppCH 2 pA (A), ppCH 2 ppA (B) and pCH 2 pppA (C). ‘A’ represents adenosine, DMF dimethylformamide, PPh3 tri- phenylphosphine, TEA triethylammonium, and TEAB triethylammonium bicarbonate. A. Guranowski et al. Methylene analogues of adenosine 5¢-tetraphosphate FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS 831 phosphate buffer (Fig. 3A), and of pppCH 2 pA from ppCH 2 pA by the use of a more complex solvent system and methanol gradient (Fig. 3B). Integrated peaks of the products were used for calculating the reaction velo- cities. As shown in Table 1, the yellow lupin p 4 A hydrolase and the yeast exopolyphosphatase hydrolysed pppCH 2 pA slightly more than twofold slower than p 4 A, and the human p 4 A hydrolase 125-fold slower. This result shows that (a) the p 4 N hydrolysing enzymes do not tolerate methylene modification of their substrates at the scissile P–O–P bond; (b) none of the enzymes hydrolysed the terminal phosphate residue from ppCH 2 ppA, or from ppCH 2 pCH 2 pA, in which the P–O–P bond between c and d phosphate remains unchanged; (c) the human hydrolase is sensitive to the –CH 2 – insert even in the most distant position from the reaction site, i.e. between the a- and b-phosphorus atoms in pppCH 2 pA. Thus the p 4 N hydrolysing enzymes are more stringent with respect to recognition of their substrates than the (asymmetrical)Ap 4 A hydro- lases, which cleave the P a –O–P b bridge not only in Ap 4 A [27], but also in AppCH 2 ppA, AppCF 2 ppA, and AppCCl 2 ppA [28]. There are obvious reasons why analogues with proximate methylene bridges should resist cleavage. For pCH 2 pppA, removal of the terminal phosphate (largely a dissociative process) is frustrated by the stability of the P c –C–P d bridge. For ppCH 2 ppA, its stability can be attributed, in at least part, to the impaired leaving group ability of b,c-methyleneATP. Neither of these explanations accounts for the much reduced activity of pppCH 2 pA for the human p 4 A Fig. 3. Time course of p 4 A (A) and pppCH 2 pA (B) hydrolysis catalysed by yeast exopolyphosphatase. Reaction mixtures (0.25 mL) were pre- pared and incubated as described in the Experimental procedures. Aliquots (0.05 mL) were withdrawn after the indicated time of incubation, the reaction was stopped by heating (96 °C, 3 min) and 2-lL sample subjected to HPLC on the Supelcosil LC-18-T reverse-phase column (25 cm · 4.6 mm). Satisfactory separation of ATP from p 4 A (A) was obtained by eluting the column with an isocratic system using 0.1 M KH 2 PO 4 buffer (pH 6.0), and separation of ppCH 2 pA from pppCH 2 pA (B) when the eluting system was a linear gradient (0–100%) of buffer A–buffer B, applied within 20 min at the flow rate 1.3 mLÆmin )1 [buffer A was 0.1 M KH 2 PO 4 + 0.008 M (CH 3 CH 2 CH 2 CH 2 ) 4 N + HSO 4 – , pH 6.0 and buffer B was buffer A: ⁄ methanol (70 : 30 v ⁄ v)]. Table 1. Comparison of the hydrolysis of ppppA and pppCH 2 pA by specific p 4 A-hydrolysing enzymes The velocities of conversion of the nucleoside tetraphosphates (0.5 m M) to corresponding nucleo- side triphosphates were calculated based on the HPLC profiles (exemplified in Fig. 3). For each enzyme the velocity of the pppCH 2 pA hydrolysis was related to that of the ppppA degradation. Enzyme Relative velocity of the pppCH 2 pA hydrolysis ppppA hydrolase from human placenta 0.8 ppppA hydrolase from yellow lupin seeds 45 Exopolyphosphatase from the yeast 42 Methylene analogues of adenosine 5¢-tetraphosphate A. Guranowski et al. 832 FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS hydrolase. It does not seem likely that such an iso- steric and isopolar analogue [47] of p 4 A could have a conformational bias that impairs access to the cata- lytic site of the enzyme by over 100-fold because it has the strongest affinity for the symmetrically clea- ving bacterial Ap 4 A hydrolase. This brings into focus the possibility of direct recognition of the P a –O–P b bridge by the protein, a possibility that might be explored by the synthesis and use of the imino ana- logue, pppNHpA. Do the analogues inhibit the p 4 A hydrolysing enzymes? All three p 4 A hydrolysing enzymes were tested with each of the six p 4 A analogues to see whether they inhibit normal hydrolysis of p 4 A. None of the ana- logues used at concentrations up to 0.5 mm retarded the conversion of p 4 A(1mm) into ATP. This unex- pected result suggests that the active sites of these three enzymes recognize and bind only nucleotides with tetraphosphate chains having intact P–O–P brid- ges, even though all of the analogues are formally isopolar and isosteric to p 4 A [47]. In this regard, it is noteworthy that recently solved structures for dUMPNPP in complex with dUTP hydrolases from Escherichia coli [48] and Mycobacterium tuberculosis [49] show a key hydrogen bond from a conserved serine hydroxyl to the imino bridge in the catalyti- cally active complex, whereas the complex between the methylene analogue dUMPCPP and the human enzyme, which cannot form such a hydrogen bond, is folded into an inactive conformation [J A Tainer, personal communication]. The methylene analogues of p 4 A as inhibitors of the (asymmetrical)Ap 4 A hydrolases Adenosine tetraphosphate itself has been known for a long time as an effective competitive inhibitor of the (asymmetrical)Ap 4 A hydrolases. Examples of the reported inhibition constants are 48 nm for the rat liver enzyme [50], 30 nm for the enzyme from Ehrlich ascites tumour cells [51] and 7.5 nm, the lowest value reported to date, for the enzyme from firefly lanterns [52]. Owing to such low K i values, this nucleotide has been used for the elution of the (asymmetrical)Ap 4 A hydrolases adsorbed to dye–ligand affinity columns as homogen- eous proteins [53,54]. We tested all six methylene and chloromethylene p 4 A analogues as potential inhibitors of two (asymmetrical)Ap 4 A hydrolases, from human and from narrow-leafed lupin, and the results are sum- marized in Table 2. Of all the analogues, ppCH 2 ppA and pCH 2 pppA appear to be the strongest inhibitors of both the human and plant enzymes. The K i values esti- mated for the human enzyme, 1.6 and 2.3 nm, respect- ively, were over 30- and 20-fold lower than the K i estimated for the same enzyme for p 4 A (50 nm). More- over, these values are five and three times smaller than the lowest K i estimated yet reported (7.5 nm) for the reaction of Ap 4 A hydrolysis catalysed by the firefly enzyme [52]. Significantly, the analogue, pppCH 2 pA, with its methylene bridge in the position most distant from the reaction site, is  100-fold less potent an inhibitor than ppCH 2 ppA. Two analogues having two methylene bridges are generally poorer inhibitors than those that possess a single methylene group. In every cases, however, the K i values were below the K m values for the Ap 4 A substrate (1 lm for the lupin and 2 lm for the human hydrolase). Finally, the analogue with the bulkiest modification, the dichloromethylene groups, was a rather poor inhibitor with K i values exceeding the K m values for Ap 4 A by some 20–50-fold. Both p 4 A and its two strongly binding methylene ana- logues inhibited the lupin enzyme 8–20-fold less effect- ively than they inhibit the human enzyme. The differential recognition of the ligands by these two hydrolases may relate to structural differences within the substrate-binding sites seen in the recently estab- lished three-dimensional structures of the lupin Ap 4 A hydrolase [55] and the human enzyme [56]. The stronger inhibition of the human enzyme by p 4 A and its analogues may be explained by the more restric- ted space in the substrate-binding cleft in the lupin enzyme. Table 2. Analogues of p 4 A as inhibitors of (asymmetrical) Ap 4 A hydrolases. The K m values for Ap 4 A estimated for the human and lupin enzyme were 2 lm (this study) and 1 lm [35], respectively. The K i values are means of three independent estimations; stand- ard errors did not exceed 20%. For details of assays see Experi- mental procedures. Human Narrow-leafed lupin (Lupinus angustifolius) ppppA 0.05 0.40 pppCH 2 pA 0.18 0.36 ppCH 2 ppA 0.0016 0.030 pCH 2 pppA 0.0023 0.034 ppCH 2 pCH 2 pA 1.3 0.07 pCH 2 ppCH 2 pA 0.25 0.62 pCCl 2 pCCl 2 ppA 40 53 ppppRib 10 n.d. ppCH 2 ppRib 0.16 n.d. pppA 16 n.d. pCH 2 ppA 2 n.d. A. Guranowski et al. Methylene analogues of adenosine 5¢-tetraphosphate FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS 833 The newly discovered ATP N-glycosidase [38] allowed us to generate the depurinated derivatives of p 4 A and of the best inhibitor analogue, ppCH 2 ppA, and evaluate the two polyphosphorylated riboses obtained as inhibi- tors of the human (asymmetrical)Ap 4 A hydrolase. It emerged that p 4 Ribose is 200 times weaker an inhibitor than p 4 A, whereas ppCH 2 ppRibose is 100 times weaker than ppCH 2 ppA. Finally, therefore, we compared the inhibition of the human recombinant Ap 4 A hydrolase by p 4 A and ppCH 2 ppA with that by ATP (p 3 A) and pCH 2 ppA, both compounds truncated by one phos- phate (and one negative charge) relative to the nucleo- side tetraphosphates. Both ATP and its b,c-methylene analogue were definitively weaker inhibitors than their d-phosphate homologues. Altogether, it is evident that both the adenine ring and the length of the polyphos- phate chain contribute to the strength of binding of the mononucleoside polyphosphates by the (asymmetrical) Ap 4 A hydrolases, whereas a single methylene bridge, preferably at or adjacent to the P–O–P reaction site, potentiates the binding. Because ppCH 2 ppA and pCH 2 pppA are the strongest inhibitors of the asymmet- rically acting Ap 4 A hydrolases ever reported and they are not degraded, in marked contrast to p 4 A which is both an inhibitor and a slow substrate for these enzymes [57,58], they clearly have excellent potential to serve as ‘true inhibitors’ and be valuable tools in biochemical and physiological studies, e.g. on nucleotide receptors. The methylene analogues of p 4 A as inhibitors of the (symmetrical) Ap 4 A hydrolase from Escherichia coli This Co 2+ -dependent enzyme was shown to hydrolyse p 4 A slowly, within a range of substrates from which it always liberates ADP as one of the reaction products [37]. The p 4 A analogues studied here are not substrates for this enzyme. However, as shown in Table 3, all act as inhibitors, albeit relatively moderate ones taking into account their inhibition of the asymmetrically act- ing Ap 4 A hydrolases. Adenosine tetraphosphate itself inhibited the enzyme with K i threefold lower than the K m for Ap 4 A (27 lm). The lowest K i value was estima- ted for pppCH 2 pA (6.7 lm) and the highest values were for ppCH 2 pCH 2 pA and pCH 2 ppCH 2 pA, 20 and 34 lm, respectively. Conclusion The data presented here show the potential usefulness of certain p 4 A analogues for the further study of the metabolism of mononucleoside polyphosphates and dinucleoside polyphosphates as well as of the function- ing of different purine ⁄ nucleotide receptors. In partic- ular, they have shown a remarkable selectivity in their behaviour as inhibitors for enzymes having super- ficially related functions as nucleoside polyphosphate hydrolases as well as showing nanomolar activity against selected enzymes. This new group of nucleotide analogues complements a different set of synthetic nucleotides, the adenosine- phosphorothioylated and adenosine-phosphorylated polyols, which has recently been proved to inhibit sym- metrically acting bacterial Ap 4 A hydrolases particularly strongly, with K i values as low as 40 nm [59]. These new, nonhydrolysable p 4 A nucleotide analogues are promis- ing tools for those who would like specifically to inhibit the asymmetrically acting Ap 4 A hydrolases. In partic- ular, they should help in structural studies of these enzymes [55,56,60]. The apparent lack of inhibition of the p 4 A hydrolysing enzymes by the methylene and chloromethylene analogues of p 4 A further challenges chemists to create other types of p 4 A analogues that may need to reach beyond the isopolar–isosteric princi- ples that have governed their design for 25 years [47]. Experimental procedures Enzymes Adenosine 5¢-tetraphosphate phosphohydrolase was obtained from yellow lupin seeds [32] and the recombinant exopolyphosphatase from yeast (S. cerevisiae) [34] was kindly donated by Dr Sh. Liu (Stanford University, CA). Adenosine 5¢-tetraphosphate phosphohydrolase from human placenta [33] was partially purified according to the following procedure. The placenta extract was fractionated with ammonium sulfate and the protein precipitated between 30 and 50% of saturation was subjected to ion-exchange chro- matography on a DEAE-Sephacel column. The enzyme was eluted with a 0–0.5 m KCl gradient, concentrated and chro- matographed on a Sephadex G-100 column from which it Table 3. Analogues of p 4 A as inhibitors of (symmetrical) Ap 4 A hydrolase from Escherichia coli.TheK m value for Ap 4 A estimated for the bacterial enzyme was 25 l M [36]. K i values are means of three independent determinations; standard errors do not exceed 15%. For details of assays see Experimental procedures. Compound K i (lM) ppppA 10.5 pppCH 2 pA 6.7 ppCH 2 ppA 16.2 pCH 2 pppA 21.8 ppCH 2 pCH 2 pA 20.0 pCH 2 ppCH 2 pA 34.0 pCCl 2 pCCl 2 ppA 8.3 Methylene analogues of adenosine 5¢-tetraphosphate A. Guranowski et al. 834 FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS eluted as a protein with molecular mass around 84 kDa. This preparation was free of any competing activity and was used for the studies of the p 4 A analogues. The recombinant human (asymmetrical)Ap 4 A hydrolase was kindly donated by Professor A. G. McLennan (University of Liverpool, UK) and the enzyme from narrow-leafed lupin from Drs D. Maksel and K. Gayler (University of Melbourne, Australia). We also used an extract from the marine sponge Axinella polypoides that contained an ATP N-glycosidase [38]. This unusual hydrolase is able to depurinate p 4 A and ppCH 2 ppA, giving d-ribose 5-O-tetraphosphate and its corresponding b,c-methylene analogue, respectively. (The sponge extract was kindly donated by Dr T. Reintamm, Tallinn, Estonia.) Chemical synthesis of p 4 A analogues Analogues with one methylene bridge Adenosine 5¢-methylenebisphosphonate was obtained by regioselective 5¢-phosphonylation of adenosine with methyl- enebis(dichlorophosphonate) using recent methodology [39]. The product was converted into the imidazolidate, ImpCH 2 pA, using imidazole with triphrenylphosphone ⁄ 2,2¢-dithio-dipyridine as the condensing agent, and this intermediate coupled with pyrophosphate to give pppCH 2 pA in 80% yield. Activation of the b-phosphate group of ADP was achieved by conversion into imidazolidate, ImppA. The activated compound was reacted with a fourfold excess of the triethylammonium salt of methylenebisphosphonate in DMF to give pCH 2 pppA [31]. The rate of pyrophosphate bond formation was greatly accelerated when carried out in the presence of an eightfold excess of ZnCl 2 [40]. Similarly, AMP was converted into adenosine 5¢-phosphoroimidazoli- date, ImpA, which was efficiently coupled with the triethyl- ammonium salt of methylenebisphosphonic acid. The resulting pCH 2 ppA was again activated with imidazole to give imidazolidate ImpCH 2 ppA, and this intermediate cou- pled with triethylammonium phosphate in a ZnCl 2 -mediated reaction to give ppCH 2 ppA in 25% yield. Schemes of these syntheses are shown in Fig. 2. Reaction mixtures were separated using DEAE-Sepha- dex 25A (triethylammonium bicarbonate gradient, pH 7.9) and ⁄ or reverse-phase HPLC (C 18 column, water ⁄ methanol gradient). HPLC analyses showed that all p 4 A analogues were at least 96% pure. Structures of all compounds syn- thesized were fully confirmed using 1 H and 31 P NMR spectroscopy and MS (see Supplementary material). Analogues with two methylene or dichloromethylene bridges Details of the procedures that led to pCCl 2 pCCl 2 ppA, ppCH 2 pCH 2 pA and pCH 2 ppCH 2 pA are given in the Sup- plementary material [41–45]. Structures of the six p 4 A analogues are presented in Fig. 1. Other chemicals Unlabelled mono- and dinucleotides were from Sigma (St. Louis, MO), and [ 3 H]Ap 4 A (740 TBqÆmol )1 ) was purchased from Moravek, Biochemicals (Brea, CA). ppppRibose and ppCH 2 ppRibose were obtained enzy- matically by incubating p 4 A and ppCH 2 ppA with the sponge ATP N-glycosidase. The progress of depurination of 2 mm nucleotides in 50 mm Hepes ⁄ KOH buffer (pH 8.0) was monitored by TLC (System A) in which the liberated adenine migrated with the solvent front. After completion of the reactions, the glycosidase was heat inactivated (3 min at 96 °C) and the mixtures used directly as a source of the depurinated compounds. The highest concentration of these compounds in the inhibi- tion assays with the (asymmetrical)Ap 4 A hydrolases was 0.05 mm. Enzyme assays Each methylene analogue of p 4 A was tested as a potential substrate for the three p 4 A-hydrolysing enzymes under the conditions established earlier as optimal for p 4 A hydrolysis. The reaction mixtures (0.05 mL final volume) contained 50 mm buffer, chloride of a divalent cation, 1 mm substrate (p 4 A or its analogue) and the investigated enzyme. For the yellow lupin p 4 A hydrolase the mixture contained Hepes ⁄ KOH buffer (pH 8.2) and 5 mm MgCl 2 , for the human enzyme Hepes ⁄ KOH (pH 7.0) and 1 mm CoCl 2 , and for the yeast exopolyphosphatase sodium acetate buffer (pH 4.7) and 1 mm CoCl 2 . Incubations were carried out at 30 °C. The results were analysed either by TLC or HPLC (see below). Asymmetrically acting Ap 4 A hydrolases were assayed in a reaction mixture (0.05 mL total volume) containing 50 mm Hepes ⁄ KOH (pH 7.6), 0.02 mm dithiothreitol, 5 mm MgCl 2 , 0.05 mm [ 3 H]Ap 4 A (300 000 c.p.m.), various con- centrations of p 4 A or its analogue and a rate-limiting quan- tity of enzyme ( 0.3 mU). For assaying the symmetrically acting Ap 4 A hydrolase from E. coli,5mm MgCl 2 was replaced with 0.1 mm CoCl 2 . Incubations were carried out at 30 °C. To estimate reaction rates, 0.005 mL aliquots were spotted on to TLC plates (aluminium plates precoated with silica gel containing fluorescent indicator; Merck cat. no. 5554), usually after 6, 12, 18 and 24 min of incubation. Unlabelled standards of the product [ATP for (asymmetri- cal)Ap 4 A hydrolases and ADP for (symmetrical)Ap 4 A hydrolase] were applied at the origin, and plates were devel- oped for 90 min in dioxane ⁄ ammonia ⁄ water (6 : 1 : 4 v ⁄ v ⁄ v). Spots of the products, visualized under short-wave UV light, were excised, immersed in scintillation cocktail, A. Guranowski et al. Methylene analogues of adenosine 5¢-tetraphosphate FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS 835 and the radioactivity measured. K i values were calculated according to the method of Dixon and Webb [46] from the slopes of plots v ⁄ v i against [I] (where v and v i are velocities in the absence and presence of inhibitor, respectively, and [I] is the inhibitor concentration), where slope ¼ K m ⁄ K i (1 ⁄ K m + S). Chromatographic systems Analyses of the hydrolysis of p 4 A or its analogues to their corresponding NTPs were performed on silica gel TLC plates developed in dioxane ⁄ ammonia ⁄ water (6 : 1 : 6 v ⁄ v ⁄ v) (System A). 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Syntheses of the p 4 A analogues with two halo ⁄ methylene bridges: General remarks on preparation of the precursors of p 4 A analogues. Synthesis of isopropyl bis(diethyl phosphonodichloro- methyl)phosphinate, pCCl 2 pCCl 2 p pentaester. Synthesis of bis(phosphonodichloromethyl)phosphi- nic acid, pCCl 2 pCCl 2 p free acid. Synthesis of adenosine-5’-[b,c,c,d-bis(dichlorometh- ylene)]tetraphosphate, pCCl 2 pCCl 2 ppA. Synthesis of a,b;b,c-bis(methylene)-ATP, tris(triethylammonium) salt, pCH 2 pCH 2 pA. Synthesis of adenosine-5’-[a,b,b, c-bis(methylene)]- tetraphosphate, ppCH 2 pCH 2 pA. Synthesis of adenosine-5’-[a,b;c,d-bis(methylene)]- tetraphosphate, pCH 2 ppCH 2 pA. Methylene analogues of adenosine 5¢-tetraphosphate A. Guranowski et al. 838 FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS . Methylene analogues of adenosine 5¢-tetraphosphate Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases Andrzej. Specific synthesis of adenosine (5¢)tetraphospho (5¢)nucleoside and adenosine (5¢)oligophospho (5¢ )adenosine (n>4) Methylene analogues of adenosine 5¢-tetraphosphate